Oncolytic therapy is a fast-developing cancer treatment field based on the promising clinical performance from the selective tumor cell killing and induction of systemic antitumor immunity. The virotherapy efficacy, however, is strongly hindered by the limited virus propagation and negative immune regulation in the tumor microenvironments. To enhance the antitumor activity, we developed injectable pH-degradable PVA microgels encapsulated with oncolytic adenovirus (OA) by microfluidics for localized OA delivery and cancer treatments. PVA microgels were tailored with an OA encapsulation efficiency of 68% and exhibited a pH-dependent OA release as the microgel degradation at mildly acidic conditions. PVA microgels mediated fast viral release and increased replication in HEK293T and A549 cells at a lower pH, and the replication efficiency could be further reinforced by co-loading with one BET bromodomain inhibitor JQ1, inducing significant cytotoxicity against A549 cells. An in vivo study revealed that OA release was highly located at the tumor tissue assisted by PVA microgels, and the OA infection was also enhanced with the addition of JQ1 treatment, meanwhile greatly inhibiting the PD-L1 expression to overcome the immune suppression. OA/JQ1 co-encapsulated injectable microgels exhibited a superior in vivo antitumor activity on the A549 lung tumor-bearing mice by the combination of inhibited proliferation, amplified oncolysis, and potential immune regulation.